There are more than 50,000 reproductive age women who are exposed to sterilizing chemotherapy and radiotherapy annually in the U.S. alone and thousands lose their ovarian function due to gynecologic surgery. Cryopreservation of ovarian tissue prior to initiation of cancer therapy, followed by autologous transplantation or in vitro maturation and in vitro fertilization after remission, could provide a means of protecting and replacing gonadal function and fertility. The destructive effects of extracellular ice formation caused by conventional cryopreservation by freezing are the major obstacle for cryopreservation of ovarian tissues. It is now well established that long-term storage of complex multicellular tissues, the integrity of which is compromised by ice formation, will best be achieved using techniques that minimize, or avoid the formation of ice. We have successfully developed vitrification method in blood vessel and articular cartilage models. The goal of this study is to investigate the effects of vitrification on structural and functional viability of ovaries compared with a conventional freezing cryopreservation method. The criteria for determining feasibility of vitrification for preserving ovaries will be (1) the demonstration of freedom from extracellular and intracellular ice formation in vitrified specimens, and (2) statistically significant differences in cell viability and follicle survival rates between ice-free cryopreserved and conventionally frozen ovaries. To our knowledge, vitrification has not previously been used for storage of large segment ovaries. This study will advance the commercial application of cryobiology in reproductive medicine. This research project is the first step towards establishment of an ovary bank for the processing, storage and distribution of the cryopreserved ovarian tissue for clinical use. Following successful completion of the aim of this Phase I SBIR proposal, several issues must be addressed in a subsequent Phase II SBIR submission. These include (1) optimization of the vitrification solutions and protocols for banking of ovaries including the development of effective methods for transportation of vitrified ovaries, (2) further investigation of the effects of cryopreservation variables upon follicle growth after in vitro maturation, and (3) in vivo evaluation of cryopreserved ovaries in a large animal (sheep) model. [unreadable] [unreadable]